Vaccines to protect against viral infections have been effectively used to reduce the incidence of human disease. One of the most successful technologies for viral vaccines is to immunize animals or humans with a weakened or attenuated strain of the virus (a “live, attenuated virus”). Due to limited replication after immunization, the attenuated strain does not cause disease. However, the limited viral replication is sufficient to express the full repertoire of viral antigens and generates potent and long-lasting immune responses to the virus. Thus, upon subsequent exposure to a pathogenic strain of the virus, the immunized individual is protected from disease. These live, attenuated viral vaccines are among the most successful vaccines used in public health.
Ten of the sixteen viral vaccines approved for sale in the U.S. are live, attenuated viruses. Highly successful live viral vaccines include the yellow fever 17D virus, Sabin poliovirus types 1, 2 and 3, measles, mumps, rubella, varicella and vaccinia viruses. Use of the vaccinia virus vaccine to control smallpox outbreaks led to the first and only eradication of a human disease. The Sabin poliovirus vaccine has helped prevent crippling disease throughout the world and is being used in the efforts to eradicate polio. Childhood vaccination with measles, mumps, rubella and varicella vaccines prevent millions of deaths and illnesses internationally.
Recent technical advances, such as reassortment, reverse genetics and cold adaptation, have led to the licensure of live, attenuated viruses for influenza and rotavirus. A number of live, viral vaccines developed with recombinant DNA technologies are in human clinical testing, including vaccines for West Nile disease, dengue fever, malaria, tuberculosis and HIV. These recombinant viral vaccines rely on manipulation of well-characterized attenuated viral vaccines, such as adenovirus, vaccinia virus, yellow fever 17D or the dengue virus, DEN-2 PDK-53. The safe, attenuated viruses are genetically engineered to express protective antigens for other viral or bacterial pathogens. Several recombinant viral vaccines have been approved for animal use, including a canarypox/feline leukemia recombinant virus, a canarypox/canine distemper recombinant virus, a canarypox/West Nile recombinant virus and a yellow fever/West Nile recombinant virus. As a group, the live attenuated virus vaccines are amongst the most successful medical interventions in human history, second only to the advent of antibiotics and hold the promise to improve public health throughout the world.
In order for live, attenuated viral vaccines to be effective, they must be capable of replicating after immunization. Thus, any factors that inactivate the virus can cripple the vaccine. For example, widespread distribution and use of the smallpox vaccine prior to World War II was limited because the virus was inactivated after only a few days at ambient temperatures. In the 1920s, French scientists demonstration that freeze-dried vaccine provided long term stability and techniques for large-scale manufacture of freeze-dried vaccine were developed in the 1940s (see for example Collier 1955). In addition to freeze-drying, various additives have been identified that can help stabilize the viruses in live, attenuated viral vaccines (See for example Burke, Hsu et al 1999). These stabilizers typically include one or more of the following components: divalent cations, buffered salt solutions, chelators, urea, sugars (e.g. sucrose, lactose, trehalose), polyols (e.g., glycerol, mannitol, sorbitol, polyethylene glycol), amino acids, protein hydrolystates (e.g. casein hydrolysate, lactalbumin hydrolysate, peptone), proteins (e.g. gelatin, human serum albumin) or polymers (e.g. dextran).
However, even with these stabilizing agents, many of the commonly used vaccines still require refrigeration for stabilization. Other commonly used vaccines are sensitive to temperature extremes; either excessive heat or accidental freezing can inactivate the vaccine. Maintaining this “cold chain” throughout distribution is particularly difficult in the developing world. Thus, there remains a need for improving the stability of both existing and newly developed live, attenuated viral vaccines.
Flaviviruses are amongst the most labile viruses. They are enveloped viruses with a RNA genome of approximately 11,000 bases. Most of the flaviviruses are transmitted by an arthropod vector, commonly mosquitoes. There are over 70 different flaviviruses that are grouped into three major categories based on serology: the dengue group, the Japanese encephalitis group and the yellow fever group. Amongst the known flaviviruses, 40 are transmitted by mosquitoes, 16 are transmitted by ticks and 18 viruses have no identified insect vector. Thus, most flaviviruses have evolved to replicate in both their arthropod vector and their vertebrate host species (often birds or mammals). Expanding urbanization, worldwide travel and environmental changes (such as deforestation or rain patterns) have lead to the emergence of several flaviviruses as threats to human public health. Such viruses include, but are not limited to, yellow fever virus, the dengue viruses, West Nile virus, Japanese encephalitis virus, and tick-borne encephalitis viruses.
Through intensive mosquito control and vaccination efforts, yellow fever was eliminated from much of North, Central and South America, the Caribbean and Europe. However, in the last 20 years, the number of countries reporting cases has increased. Yellow fever virus is now endemic in major portions of Africa and South America and some Caribbean islands. The World Health Organization (WHO) estimates that 200,000 cases of yellow fever occur annually leading to 30,000 deaths. Since World War II, dengue flaviviruses have spread to tropical and subtropical regions throughout the world and now threaten over 3.5 billion people, about half of the world's population. The WHO estimates that 50-100 million cases of dengue fever occur annually. 500,000 of these are the more severe, life-threatening form of the disease, termed dengue hemorrhagic fever, that leads to more than 25,000 deaths per year. A particularly virulent form of West Nile virus was introduced into the Western hemisphere, presumably by travel, in New York in 1999. The mosquito-transmitted virus infected birds as the primary host, but also caused disease and mortality in humans and horses. West Nile virus spread throughout the United States and into Canada and Mexico. Since its introduction, West Nile virus has caused over 20,000 reported cases of West Nile disease leading to 950 deaths in the United States. Japanese encephalitis virus causes 30,000 to 50,000 cases of neurological disease annually, primarily in eastern and southern Asia. 25-30% of the reported cases are fatal. The tick-borne encephalitis viruses are endemic to parts of Europe and Asia and continue to cause episodic outbreaks affecting thousands of individuals. Related viruses with more limited geographical spread include Kunjin virus (a close relative of West Nile) and Murray Valley encephalitis virus in Australia and New Guinea, St. Louis encephalitis virus in North and South America, the Usutu, Koutango, and Yaonde viruses in Africa, and Cacipacore virus in South American.
Live, attenuated viral vaccines have been developed that are safe and protect against flavivirus diseases, such as yellow fever and Japanese encephalitis. The live, attenuated viral vaccine, 17D, has been widely used to prevent yellow fever. The current flavivirus vaccines are lyophilized in the presence of stabilizers. Nonetheless, the vaccines require storage and shipment at 2-8° C., a requirement that is difficult to achieve in the developing world and more remote regions of developed nations. Furthermore, upon reconstitution, the vaccines rapidly lose potency even when stored at 2-8° C.
The measles vaccine is another example of a labile attenuated virus that is used worldwide to prevent disease. Measles virus is an enveloped, non-segmented negative strand RNA virus of the Paramyxovirus family. Measles is a highly contagious, seasonal disease that can affect virtually every child before puberty in the absence of vaccination. In developing countries, mortality rates in measles-infected children can by as high as 2 to 15%. Indeed, despite efforts to institute worldwide immunization, measles still causes greater than 7,000 deaths in children per year. The measles vaccine is a live, attenuated virus that is manufactured in primary chicken fibroblast cells. The vaccine is stabilized with gelatin and sorbitol and is then lyophilized. The stabilized, lyophilized vaccine has a shelf life of 2 years or more if stored at 2 to 8° C. However, the lyophilized vaccine still requires a cold chain that is difficult to maintain in the developing world. Furthermore, upon reconstitution, the vaccine loses 50% of its potency within 1 hour at room temperature (20 to 25° C.).
Thus, a need exists in the art for improved vaccine formulations.